High fat:low carbohydrate relative to high carbohydrate:low fat feeding is associated with a marked reduction in insulin's ability to stimulate glucose transport in the isolated rat adipose cell. Since insulin appears to enhance glucose transport by triggering a rapid and reversible translocation of glucose transport systems from a large intracellular pool to the plasma membrane, the effects of high fat:low carbohydrate feeding on this pool and the translocation process in the isolated rat adipose cell have been examined. With high fat:low carbohydrate feeding, insulin's ability to stimulate 3-0-methylglucose transport in intact cells and to increase the concentration of glucose transport systems in intact cells and to increase the concentration of glucose transport systems in the plasma membrane fraction prepared from these cells is decreased by 55% and 41%, respectively. At the same time, the basal concentration of glucose transport systems in the low-density microsomal membrane fraction prepared from cells of the high fat:low carbohydrate-fed rats is decreased by 47%. Thus, the reduced glucose transport response of the rat adipose cell to insulin with high fat:low carbohydrate feeding appears to be the consequence of a relative depletion of glucose transport systems in the intracellular pool.